THE PHYSIOLOGY OF IGFBP DEGRADING PROTEINASES IN BONE

描述（申请人提供）：胰岛素样生长因子（IGFs）是成骨细胞生长和分化的重要介质；然而，六种高亲和力的IGF结合蛋白（igfbp 1-6）拮抗它们与细胞表面1型IGF受体的结合。骨细胞产生的蛋白酶可以将igfbp降解为低亲和力的片段，从而使IGF-IGF受体相互作用发生。我们发现妊娠相关血浆蛋白a （PAPP-A）是IGFBP-4蛋白酶，MMP-13（胶原酶-3）是IGFBP-5蛋白酶，两者都是由成骨细胞系MC3T3-E1产生的。因此，这些igfbp的蛋白水解过程可能会在成骨细胞形成过程中调节IGF的作用。我们已经创建了抗蛋白水解的IGFBP-4和IGFBP-5的重组突变形式，并利用这些结构建立了稳定转染的MC3T3-E1细胞系，这些细胞系过度表达了蛋白酶抗性IGFBP突变体。在此，我们建议使用这些试剂来探索IGFBP蛋白水解如何影响成骨细胞的生长和分化。我们将比较蛋白酶抗性形式的IGFBP-4或-5与可切割形式相比如何影响成骨细胞的形成，以及抑制或下调igfbp降解蛋白酶（即PAPP-A和MMP-13）是否影响这一过程。在体内，我们将研究蛋白酶抗性或敏感形式的IGFBP-4或-5如何改变新骨的形成，以及PAPP-A是否可以克服转基因过量生产IGFBP-4对新骨形成的骨抑制作用。为了探索MMP-13如何介导甲状旁腺激素对骨细胞的合成代谢影响，我们将确定PTH介导的MMP-13上调的钝化如何改变体外成骨细胞中IGF的作用。我们还将探讨抗MMP-13形式的IGFBP-5如何影响成骨细胞培养中PTH的作用。体内研究将确定全身PTH给药是否会改变骨中的IGF-IGFBP-IGF蛋白酶轴，以及MMP-13的缺失是否会影响PTH和igf介导的新骨形成。总之，这些研究将为控制IGF在骨中的生物利用度的机制，以及骨形成的上游介质（如甲状旁腺激素）如何通过调节IGF的作用来影响成骨细胞的形成提供新的见解。

DESCRIPTION (provided by applicant): Insulin-like growth factors (IGFs) are important mediators of growth and differentiation of osteoblasts; however, six high-affinity IGF-binding proteins (IGFBPs 1-6) antagonize their binding to cell surface type-1 IGF receptors. Proteases produced by bone cells can degrade IGFBPs into low-affinity fragments, allowing for IGF-IGF receptor interactions to occur. We have identified pregnancy-associated plasma protein-A (PAPP-A) as an IGFBP-4 proteinase and MMP-13 (collagenase-3) as an IGFBP-5 proteinase, and both are produced by the osteoblast cell line, MC3T3-E1. Thus, proteolytic processing of these IGFBPs may modulate IGF action during osteoblastogenesis. We have created recombinant mutant forms of IGFBP-4 and IGFBP-5 that are resistant to proteolysis, and have used these constructs to establish stably transfected MC3T3-E1 cell lines overexpressing protease resistant IGFBP mutants. Herein, we propose to use these reagents in order to explore how IGFBP proteolysis influences osteoblast growth and differentiation. We will compare how protease-resistant forms of IGFBP-4 or -5 impact osteoblastogenesis compared to cleavable forms, and if inhibiting or down-regulating IGFBP-degrading proteinases (i.e., PAPP-A and MMP-13) influences this process. In vivo, we will examine how protease-resistant or sensitive forms of IGFBP-4 or -5 alter new bone formation, and if PAPP-A can overcome osteoinhibitory effects of transgenic overproduction of IGFBP-4 on new bone formation. To explore how MMP-13 may mediate anabolic affects of PTH on bone cells, we will determine how blunting of PTH-mediated up-regulation of MMP-13 may alter IGF action in osteoblasts in vitro. We will also explore how MMP-13 resistant forms of IGFBP-5 impact PTH actions on osteoblast cultures. In vivo studies will determine if systemic PTH administration alters the IGF-IGFBP-IGF protease axis in bone and if deletion of MMP-13 affects PTH and IGF-mediated new bone formation. Together, these studies will provide new insights into mechanisms controlling IGF bioavailability in bone, and how up-stream mediators of bone formation, such as PTH, may exert their affects on osteoblastogenesis through regulating IGF action.